1. Field of the Invention
Embodiments of the invention disclosed herein relate generally to compositions and methods for altering hepatocyte growth factor activity or c-Met receptor activity. Certain aspects relate to the diagnosis, prevention or treatment of, as well as to general therapy of subjects having, suspected of having, or susceptible to, a condition associated with c-Met receptor dysregulation. C-Met receptor dysregulation may be a condition in which underactivity, overactivity or improper activity of a c-Met cellular or molecular event is present, including obesity or a condition associated with obesity, a hyperproliferative disorder, a condition characterized by abnormal angiogenesis, or alternatively, a condition characterized by vascular insufficiency such as may benefit from increased angiogenesis. Embodiments disclosed herein further relate to methods for identifying or modifying compounds useful for the diagnosis, prevention or treatment of such conditions associated with c-Met receptor dysregulation.
2. Background of the Invention
The classic renin-angiotensin system regulates cardiovascular function including blood pressure, electrolyte balance, reproduction, and may play a role in other physiological processes, including atherosclerosis. These angiotensin-mediated effects are believed primarily to operate through angiotensin (AT) receptors identified as AT1 and AT2 receptors. Renin, through its proteolytic activity, first cleaves the angiotensinogen precursor polypeptide to form angiotensin I. Next, angiotensin converting enzyme (ACE) enzymatically converts angiotensin I to angiotensin II; ACE has been detected in a variety of tissues including brain, kidney, adrenal glands, vasculature, heart and ovaries. ACE-generated angiotensin II (AT2 or AT2) is subsequently cleaved (by aminopeptidase A) to form angiotensin III, which is cleaved by aminopeptidases N, M and/or B to form angiotensin IV (Val-Tyr-Ile-His-Pro-Phe, [SEQ ID NO:33]), (Mustafa et al., J. Renin Angiotens, Aldoster. Syst. 2(4):205-210, (2001), Thomas et al., Int. J. Biochem. & Cell Biol. 35: 774-779 (2003): McKinley et al., Int. J. Biochem. & Cell Biol. 35: 901-918 (2003).)
Angiotensin IV (AT4 or AT4) has been shown to play a role in regulating disparate biological activities including blood flow, cognitive function, neuronal development, inflammation and behavior (Wright et al., Prog. Neurobiol. 72: 263-293 (2004); Kamar et al., Regul. Pept. 68: 131-138 (1997).) AT4 is believed to exert its biological effects through interaction with a cell surface receptor identified as the AT4 receptor (AT(4)R), which is also known as the insulin-regulated membrane aminopeptidase (IRAP) (e.g., Chai et al., 2004 Cell. Mol. Life. Sci. 61:2728; Esteban et al., 2005 Circ. Res. 96:965; Albiston et al., 2001 J. Biol. Chem. 276:48623). AT(4)R/IRAP is a type II (see, e.g., Parks, 1996 J. Biol. Chem. 271:7187) integral membrane-spanning protein having aminopeptidase activity.
Many of the observed biological characteristics of the AT4 system (e.g., the existence of both agonist and antagonist AT4 ligands), however, have been difficult to reconcile with the TRAP model for the AT4 receptor. This discrepancy suggested that another unidentified protein(s) might be responsible for the action of AT4 receptor ligands (Harding et al., 1994 Kidney Int. 46:1510; Wright et al., 2004 Prog. Neurobiol. 72:263). The molecular identity of the target AT4 receptor that mediates a number of AT4 biological activities has, however, remained elusive.
Hepatocyte growth factor (HGF, e.g., 1991 Proc. Nat. Acad. Sci. USA 88:7001; Donate et al., 1994 Prot. Sci. 3:2378; GenBank Accession No. AAA64239 [SEQ ID NO:83], M73239.1 (728 amino acids)), also known as scatter factor (SF), is a noncovalent homodimeric polypeptide growth factor, assembled from naturally occurring monomeric HGF polypeptide subunits, that induces cell motility and cell proliferation, which may lead to normal processes of angiogenesis, or abnormal processes of tumor development or metastasis. HGF functions by binding to its cell surface receptor, c-Met, which is a receptor protein tyrosine kinase and a protooncogene product.
The c-Met receptor is a heterodimer composed of an alpha and beta chain (Maggiora et al., J. Cell Physiol. 173:183-186, (1997), Christensen et al., Can. Lett. 225: 1-26, (2005)). The c-Met receptor is enriched on vascular endothelial cells where it mediates the regulation of angiogenesis (Rosen et al., 1997 EXS 79:193). For instance, NK4, a large molecule c-Met inhibitor, has been shown previously to inhibit angiogenesis (Kuba et al., 2000 Cancer Res. 60:6737).
Upon activation, as may result from ligand engagement, the c-Met polypeptide (e.g., hepatocyte growth factor-receptor, HGF-R, also known as scatter factor receptor, SF-R, GenBank Acc. No. AAA59591, [SEQ ID NO:84]) induces mitogenic, motogenic and morphogenic responses by recruiting a number of signaling and docking molecules, and has been implicated in the phosphorylation of cell junction proteins (e.g., Zhang et al., 2003 J. Cell Biochem. 88:408; Miao et al., 2003 J. Cell Biol. 162:1281; Berdichevsky et al., 1994 J. Cell Sci. 107:3557). Ligand induced activation of c-Met by HGF/SF leads to the autophosphorylation of specific tyrosine residues within the c-Met receptor protein tyrosine kinase (PTK) domain (Furge et al. (2000) Oncogene 19, 5582-5589; Weidner et al. (1995) Proc Natl Acad Sci USA 92, 2597-2601) and to the association of various signaling proteins (e.g., Naldini et al., 1991 Mol. Cell. Biol. 1250:1085). A significant event in c-Met signaling is the association with the c-Met receptor of growth factor receptor bound protein 2, Grb2 associated binder (Gab1), a multi-functional scaffolding adapter (Birchmeier et al., 2003 Nat. Rev. Mol. Cell. Biol. 4:915). Gab1 association provides c-Met with multiple docking sites for a variety of intracellular signal transducers (Trusolino et al., 2002 Nat. Rev. Cancer 2:289).
Following activation by HGF/SF, c-Met is able to exert a variety of effects by recruiting docking and signaling molecules. Phosphorylation of the tyrosine residues in the activation loop of the PTK domain potentiates the intrinsic kinase activity of Met, whereas phosphorylation of the two docking site tyrosine residues (Tyr1349, Tyr1356) allows for the recruitment of adaptor molecules including Grb2, SHC and Gab1 and signaling enzymes including phosphotidylinositol 3-kinase (PI3K), phospholipase C.gamma. (PLC-.gamma.), the PTK src, the protein tyrosine phosphatase SHP2, as well as the transcription factor STAT3 (reviewed in Furge et al. (2000) Oncogene 19, 5582-5589).
The binding of HGF to the cell surface c-Met receptor thus results in multiple cell-signaling events that promote cell survival, cell proliferation, cell motility, disruption of the extracellular matrix (ECM), cell morphogenesis, angiogenesis and/or cell extravasation and colonization, for instance, as observed in tumor metastasis. (Jeffers et al., J. Mol. Med., 74: 505-513 (1996); Amicone et al., EMBO J. 16: 495-503 (1997); Matsumoto and Nakamura, Biochem. Biophys. Res. Comm 239: 639-644 (1997); Kirchhofer et al., J. Biol. Chem., 279: 39915-39924 (2004)). Disruption of normal signaling through c-Met has been implicated in certain cancers (e.g., Zhang et al., 2004 Cancer Cell 6:5; Christensen et al., 2005 Cancer Lett. 225:1-26; Ferraro et al., 2006 Oncogene 25:3689). For example, overexpression of HGF and/or of c-Met has been implicated in a number of cancers, including carcinomas, gliomas, and mesotheliomas. Particular organs affected include breast, pancreas, liver, lung, ovary, stomach, bile duct, kidney, and others, in part because of increased angiogenesis (Zbar et al., J. Urol., 151: 561-566 (1994); Date et al., FEBS Letters, 420:1-6 (1997)). In addition, several studies have indicated that cancer cells can be a significant source of HGF within a subject (e.g., Jiang et al., One. Hemat. 53: 35-69 (2005)).
Alterations (e.g., statistically significant increases or decreases) in the activity states of a number of intracellular signaling cascades thus characterize cellular responses to HGF binding by the cell surface c-Met receptor, including biological signal transduction pathways that comprise one or more of Grb2, cortactin, Arp2/3, WASP/Wave, Rho/rac, Rock, LIMK, PI1P5-K, ERM proteins, Dia-1, MLC phosphatase, cofilin, Ptdins(4,5)P2, cadherins (including E-cadherin), MMPs, fl-catenin, p27kip1, SOS, Ras, Raf, MAPK, PI3K, NK B, src, JNK1, Bid/Bax, caspases, C-Myc, Bax, Mcl1, Bcl-w, Akt, FLICE, STATs (including STAT3), COX, ERK/paxillin, as well as others (see, e.g., Jiang et al., One. Hemat. 53: 35-69 (2005); Alberts et al., Molecular Biology of the Cell, 4th Ed., 2002, Garland Science, N.Y.). Activation of these intracellular messenger systems can lead to changes in a cell's cytoskeleton, adhesion state and adherens junctions, cell cycle, and directional cell movement, and may also contribute to altered activity in one or more of a number of other biochemical pathways that affect cellular metabolic, catabolic, biosynthetic, respiratory, gene expression, membrane dynamic or other functions or phenotypes. Thus, such HGF-c-Met binding events may lead to or contribute to cancer development, tumor cell growth or metastasis, altered angiogenesis, or other physiologically significant outcomes.
Angiogenesis, the process of blood vessel formation, is necessary for proper wound healing and repair, as well as playing an important role during embryonic, fetal and young animal development, and continuing on to adulthood. Dysfunction in the course of angiogenic processes at any of these stages may result in certain detrimental health conditions, including, for instance, ischemic heart disease, preeclampsia, neurodegeneration, and/or respiratory distress (e.g., as the result of an inadequate or insufficient level of angiogenesis relative to the levels seen in unafflicted individuals), and also including, for example, malignant metastasis, arthritis, macular degeneration, diabetic retinopathy, ocular and inflammatory disorders, obesity, asthma, diabetes, cirrhosis, multiple sclerosis, endometriosis, AIDS, bacterial infections, and/or autoimmune diseases (e.g., as the result of an excessive or overabundant level of angiogenesis relative to the levels seen in unafflicted individuals). See, e.g., Carmeliet, Nature 438:932-936 (2005). Intervention to alter (e.g., increase or decrease in a statistically significant manner) angiogenesis in these and other conditions remains a useful but incompletely fulfilled goal.
Of the conditions relating to dysfunction in angiogenesis as described above, obesity is one in which adipose tissue formation, or adiposity, is increased to a point where it is associated with certain clinically defined health conditions or increased mortality. Although obesity is an individual clinical condition, it is increasingly viewed as a serious and growing public health problem, as excess adipose tissue is accompanied by a dramatically increased risk for the development of numerous recognized health problems, including insulin resistance and type 2 diabetes mellitus, impaired glucose tolerance, non-alcoholic fatty liver disease, dyslipidemia (characterized by elevated levels of nonesterified fatty acids (NEFAs), triglycerides, and small dense LDL particles, along with reduced levels of HDL), hypertension, coronary heart disease, increased inflammatory activity, and thrombosis (Bray J. Clin. Endocrinol. Metab. 89:2583-89 (2004); Glass and Witstum, Cell 104:503-516 (2001)). These diseases are associated primarily with an increased number of fat cells.
Other conditions associated with excess adipose tissue formation, and which may therefore be associated with obesity, relate in particular to increased fat mass, including immobility, osteoarthritis, respiratory conditions such as dyspnea and obstructive sleep apnea, and psychological problems such as depression and social stigmatization. A relationship has also been identified between obesity and the incidence of certain cancer types, such as breast cancer (Calle and Kaaks, Nat. Rev. Cancer 4:579-591 (2004); and lyengar et al., Oncogene 22:6408-6423 (2003)), in addition to endometrial, colorectal, kidney, prostate, gallbladder, pancreatic and esophageal cancers (See Vainio and Bianchini, IARC handbooks of cancer prevention. Volume 6: Weight control and physical activity. Lyon, France: IARC Press, 2002); Abu-Abid et al., J Med 33:73-86 (2002); and Giovannucci, Gastroenterology 132:2208-25 (2007)).
Angiogenesis regulates the growth and maintenance of adipose tissue, which is a highly vascularized tissue. Neovascularization is a feature of adipose tissue expansion (e.g., adipogenesis) as well as of adipose tissue maintenance, suggesting that adipose tissue viability is sustained by a constant vascular remodeling process (Dallabrida et al., Biochem. Biphys. Res. Commun. 311:563-571 (2003)). As one particular example of this phenomenon, vascular endothelial growth factor (VEGF) expression and resulting angiogenesis may augment or precipitate adipogenesis in white and brown adipose tissues (Hausman and Richardson, J. Anim. Sci. 82:925-34 (2004)).
Angiogenesis regulatory proteins may provide relevant, therapeutic targets for controlling adipose tissue formation, and in particular, angiogenesis inhibitors may be useful in reducing obesity and its associated health problems (Liu and Meydani, Nutrition Reviews 61:384-387 (2003)). For example, systemic treatment of obese mammals with anti-angiogenic agents has been shown to induce a loss of white adipose tissue (Rupnick et al. PNAS USA 99:10730-35 (2002). In particular, TNP-470, a selective inhibitor of endothelial growth and angiogenesis, and other angiogenesis inhibitors such as endostatin, angiostatin, thalomide, and leptin, have all been shown to reduce obesity in mice (Id.; and Brakenhielm et al., Circulation Research 94:1579-1588 (2004)).
While intervention strategies that target angiogenic regulatory factors would therefore appear useful for controlling adiposity, and thus obesity, systemically delivered anti-angiogenic agents do not exhibit sufficient specificity for adipose tissue to provide a practical therapeutic modality. Accordingly, there is a need in the art to identify molecular targets in angiogenic pathways that would provide increased specificity for adipose tissues.
HGF, in particular, regulates angiogenesis in adipose tissue, and thus regulates adiposity. In fact, obesity is associated with elevated levels of circulating HGF (Rehman et al., Journal of the American College of Cardiology 41:1408-13 (2003), and adipose tissue has been shown to produce HGF, thereby contributing specifically to such elevated HGF levels (Bell et al., Am J Physiol Endocrinol Metab 291:E843-E848 (2006). In addition, in vivo silencing of HGF expression in preadipocytes decreases the ability of these cells to recruit endothelial cells for angiogenesis, whereas elevated HGF expression in preadipocytes enhances endothelial cell migration into fat tissue (Bell et al., Am J Physiol Endocrinol Metab (in press, 2007). HGF is thus a potent mitogenic and angiogenic factor that is produced in human adipose tissue, and that plays a central role in adipose tissue angiogenesis.
Highlighting the relationship between obesity and cancer, as noted above, aberrant HGF expression is also implicated in many types of cancer. By way of example, a number of tumors, including melanomas, hepatomas, and carcinomas of the breast, exhibit inappropriate expression of HGF, and HGF overexpression in transgenic mice results in neoplasms of the liver, mammary gland, skeletal muscle, and melanocytes (Jabubczak et al., Mol Cell Biol 18:1275-83 (1998). Targeting of the HGF pathway, alone or in combination with standard therapies, is being considered as a means of improving current therapies in certain types of malignancies (Sattler and Salgia, Curr Oncol Rep 9:102-8 (2007)). The presence of elevated HGF expression in both obesity and certain types of cancer represents a potential therapeutic nexus between these two significant public health problems.
HGF appears to be a critical regulator of neural stem cell expansion and differentiation (Nicoleau et al., 2009) suggesting that neural as well as many types of peripheral stem cells are under the control of the HGF/c-Met system. HGF has been shown to be a potent neurotrophic factor (Ieraci et al., 2002; Kato et al., 2009) in many brain regions and to be particularly effective as a pro-survival/regenerative factor for motoneurons (Kitamura et al., 2011), hippocampal neurons (Lin and Walikonis, 2008), cerebellar granular cells (Ieraci et al., 2002), and sympathetic neurons (Maim and Klein, 1999). The neuroprotective effect of the HGF on the nigrostriatal pathway may be credited to its role in stem cell regulation (review, Nakamura et al., 2011), its effect on the development of tubular structures (Santos et al., 1993) and its general proliferative, anti-apoptotic, motogenic, and morphogenic actions on many cell types including hepatocytes and cells of epithelial origin (Bai et al. (2012). The use of HGF may thus also be applicable to a variety of neurological diseases and conditions, several of which are described in the following paragraphs.
Dementia
There are approximately 10 million diagnosed dementia patients in the United States alone and that number continues to grow every year as the population ages. The costs of treatment and care of these patients are in excess of $70 billion annually and are increasing rapidly. Unfortunately, the current treatment options for the management of dementia are severely limited and largely ineffective. The lack of treatment options for a burgeoning health problem of this magnitude necessitates that new and innovative therapeutic approaches be developed as quickly as possible.
At its core dementia results from a combination of diminished synaptic connectivity among neurons and neuronal death in the entorhinal cortex, hippocampus and neocortex. Therefore, an effective treatment would be expected to augment synaptic connectivity, protect neurons from underlying death inducers, and stimulate the replacement of lost neurons from preexisting pools of neural stem cells. These clinical endpoints advocate for the therapeutic use of neurotrophic factors, which mediate neural development, neurogenesis, neuroprotection, and synaptogenesis. Not unexpectedly neurotrophic factors have been considered as treatment options for many neurodegenerative diseases including Alzheimer's disease (Fafalios et al., 2011); Kitamura et al., 2011).
Amyotrophic Lateral Sclerosis (ALS)
ALS is a fatal rapid-onset neurodegenerative disease that is characterized by degeneration of motoneurons in the spinal cord and efferent neurons in the motor cortex and brainstem. The impact of this degeneration results in a progressive loss of muscle function culminating in total paralysis. Approximately 90% of the cases of ALS are classified as sporadic with no known etiology, while the remaining 10% appear to be familial, resulting in part from defects in copper/zinc superoxide dismutase 1 (SOD1), which leads to exaggerated oxidative stress and an unfolded protein response. The one thing that both forms of ALS have in common is that there is currently is no effective treatment.
Despite the paucity of effective treatment options, several studies have highlighted the potential benefits of using hepatocyte growth factor (HGF) as a therapeutic agent. These investigations have demonstrated that application of hepatocyte growth factor (HGF) in a murine or rat model of familial ALS significantly slows motoneuron degeneration (Fafalios et al., 2011); reduces gliosis (Kitamura et al. 2011), which contributes to the degeneration process; delays the onset of paralysis (4); and increases lifespan (Fafalios et al. 2011).
Parkinson's Disease
A treatment option long considered for many neurodegenerative diseases including Parkinson's disease (PD) has been the application of growth factors with the intention of halting disease progression, restoring lost function, or hopefully both (Rangasamy et al., 2010). However, this objective has gone largely unfulfilled at the level of clinical medicine because of limitations related to brain delivery and costs. Growth factors are universally large proteins that are both metabolically labile and too large to pass the blood-brain barrier (BBB). As such, most approaches to delivery have utilized gene therapy methods with the hope that the growth factor will be expressed in the correct location at a high enough concentration and for a long enough period to provide clinical relief. Although a number of creative and successful approaches in animal models have been employed to deliver growth factors like GDNF (Wang et al., 2011) to the brain, these methodologies are technically complex and prohibitively difficult to bring to practice with large numbers of patients.
The potential utility of HGF as a PD treatment has been highlighted in a study by Koike et al. (2006) in which an HGF plasmid injected directly into the substantia nigra (SN) resulted in localized over-expression of HGF, and acted dramatically to prevent neuronal cell death and preserve normal motor function in the 6-hydroxydopamine (6-OHDA) PD rat model. This observed neuroprotective effect of HGF on dopaminergic (DA) neurons meshes with its ability to augment the proliferation and migration of dopaminergic progenitor cells (Lan et al., 2008).
Traumatic Brain Injury (TBI)/Spinal Cord Injury
TBI often negatively impacts cognitive function and can elicit effects that range from mild, with temporary decrements in mental abilities, to severe, with prolonged and debilitating cognitive dysfunction (Kane et al., 2011). Cognitive difficulties along with other neurological deficits including: anxiety, aggressiveness, and depression result in a significantly reduced quality of life (Masel and DeWitt, 2010). With military operations concluded in Iraq and continuing in Afghanistan TBI has become the major combat injury at 28% of all combat casualties (Okie, 2005; U.S. Medicine, May 2006, Vol 42). Total estimates of military service members suffering TBIs between 2001 and 2010 range from 180,000 to 320,000 (U.S. Defense and Veterans Brain Injury Center).
Underlying TBI is physical injury to the brain resulting in decreased synaptic connectivity among neurons, loss and death of neurons, damage to cerebral blood vessels resulting in ischemic/hypoxic-induced damage, and secondary glial scaring. This loss of neurons and diminished synaptic connectivity is particularly apparent in the hippocampus (Gao et al., 2011; Zhang et al., 2011a; Zhang et al., 2011b) resulting in defective long-term potentiation (Schwarzbach et al., 2006) and cognitive deficits (e.g. Dikmen et al., 2009). The prevalence of TBI associated injuries that result in neuronal loss and decreased synaptic connectivity denote the need for therapies which support neuronal repair and/or replacement. These clinical endpoints advocate for the therapeutic use of neurotrophic factors such as HGF which mediate neural development, neurogenesis, neuroprotection, and synaptogenesis, for treating TBI. Neurotrophic factors have been considered as treatment options for TBI (Kaplan et al., 2010; Richardson et al., 2010; Qi et al., 2011). Further, recent studies have demonstrated that HGF given intrathecially reduces tissue damage and promotes functional recovery in a non-human primate spinal cord injury model when it is delivered after injury (Kitamura et al., 2011). HGF prevented glial scarring, an impediment to axon regrow, while at the same time stimulating the regrowth process.
Multiple Sclerosis
HGF derived from mesenchymal stem cells (MSCs) has recently been shown to reduce functional deficits in mouse MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE) and promote the development of oligodendrocytes and neurons resulting in enhanced neural cell development and remyelination (Bai et al., 2012). These data strongly indicate that HGF mediates functional recovery in a standard multiple sclerosis model and thus suggest that augmented HGF function may have significant therapeutic benefits in MSC multiple sclerosis patients.
Unfortunately, the direct use of HGF or any other protein neurotrophic factor as a therapeutic agent has serious limitations. For example, HGF is manufactured by recombinant methods at a very high cost, severely limiting its widespread use. With respect to neurological disorders in particular, the large size and hydrophilic character of HGF preclude blood-brain barrier permeability (BBB). Accordingly, there is a need in the art for low cost, readily administrable and yet effective compounds and compositions that are capable of specifically and selectively regulating HGF activity in vivo.